Key stress factors and parameters for batch production

optimisation of silk-elastin-like proteins in E. coli Tony Collins, João Azevedo-Silva, André da Costa, Raul Machado, Margarida Casal

Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

Conclusions

Maximum SELP production in shake flasks was obtained with terrific broth at 37ºC, pH7.0 with a liquid vol.:vessel vol. ratio of 1:10 and an agitation speed of 200 rpm.

Maximum induction is obtained at the end of the declining exponential phase with an induction period of at least 4 hours. This allows for high cell density on induction and

reduces the rate of plasmid loss after induction.

Production levels of approximately 500 mg/L were obtained after purification.

Rapid degradation of the selection agent used, plasmid instability on induction and high acetate byproduct formation are the principal factors preventing further improved

production levels with the expression system used.

We are presently investing the fed-batch approach for overcoming some of these restraints and to further increase production levels of the novel SELP.

Introduction - Silk-elastin-like proteins (SELPs) are a family of biopolymers based on the highly repetitive amino acid sequence blocks of the naturally occurring fibrous proteins silk and elastin..

- In contrast to conventional synthetic polymers, the composition, sequence and length of these biopolymers can be strictly controlled, leading to monodispersed, precisely defined

polymers which can be biosynthesised in an ecologically friendly manner, are biodegradable and biocompatible.

- SELPs combining the physicochemical and biological properties of the high tensile strength silk with highly resilient elastin allow for the fabrication of diverse materials with a high

potential for use in the pharmaceutical, regenerative medicine and materials fields, yet their development for use is restrained by their typically low production levels.

- A series of novel SELPs composed of multiple blocks of the silkworm silk consensus sequence GAGAGS in various combinations with a mutated variant (VPAVG) of the natural

mammalian elastin repetitive sequence block VPGVG have recently been prepared.

- The genes encoding the newly designed SELPs have been synthesised and the novel recombinant polymers expressed in E. coli by use of the pET25b-E. coli BL21(DE3) expression

system.

Aims - To optimise the shake flask production levels of the novel SELP (S5E9)10 by means of a comprehensive empirical approach examining all process variables (see 1 to 4 below).

- To obtain a better understanding of the factors limiting further improved recombinant protein production of shake flask cultivations with the expression system used (see 5 to 7 below).

1. Culture Medium

• Highest biomass and SELP

production were observed with rich

buffered media such as terrific broth

(TB) and super broth (SB).

• Variation of the sugar supplement in

TB, modification of its ingredient

concentrations, or use of additives

such as ammonium, various amino

acids, magnesium or NaCl did not

improve production levels further.

2. Temperature

• 37 – 42ºC allowed for highest

production levels

• Highest biomass production was

observed at 25ºC but SELP production

was low, possibly due to repression of

the T7-based expression system at this

temperature.

• Upshifts in temperature on induction

did not lead to significantly higher

production levels.

0

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0

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60

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120

LB LBM

TB TBlac

TBm

od

TBaim

SB Sbm

od

SBaim

Sben

rich

SOC

NB

S

NB

Smo

d

ZYB

ZYBb

uff

M9

Ries

Max. A

60

0n

m, M

in. p

H

SELP

Pro

du

ctio

n: %

of

Max

.

Medium SELP Prod.: % of Maximum Max. A600nm Minimum pH

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25 30 37 42 25-30 25-37 30-37 37-30 37-42

Maxu

mu

m O

D6

00

nm

SELP

Pro

du

ctio

n: %

of

Max

.

Temperature

PBP Prod. (% of Maximum) Maximum OD600nm

3. Available Oxygen

• Increasing aeration, or oxygen transfer efficiency, as

indicated by increasing flask vol. to liquid vol. ratio

and increasing agitation, correlated with improved

biomass and SELP levels up to a limit.

• At higher aeration rates toxic byproduct production

(e.g. acetate) due to the higher growth rates observed

probably interfere with production.

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0 5 10 15 20

Maxim

um

OD

60

0n

m

SELP

Pro

du

ctio

n: %

of

Max

.

Ratio of Flask Volume to Medium Volume

Flask Volume: Medium Volume Optimisation

SELP Production: % of Maximum Max. A600nm

0

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80 120 160 200 240

Maxim

um

OD

60

0n

m

SELP

Pro

du

ctio

n: %

of

Max

.

Agitation (rpm)

Agitation

SELP Production: % of Maximum Max. A600nm

Fig. 4: - Effect of

varying the agitation

rate on biomass and

SELP production..

Fig. 3 - Effect of varying

the flask volume to culture

medium volume ratio on

biomass and SELP

production.

4. Induction

• In contrast to most studies which induce

during the mid-log phase, we observed

maximum production with induction at the

end of the declining exponential phase.

• At least four hours induction is

recommended for maximum production.

0

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2 4 6 8 10 14

Maxim

um

OD

60

0n

m

SELP

Pro

du

ctio

n: %

of

Max

.

Time of Induction (hrs.)

Induction Time and Induction Period

SELP Production: 2 hrs. Induction SELP Production: 4 hrs. Induction

SELP Production: 6 hrs. Induction Maximum OD600nm

0,1

1

10

0 2 4 6 8 10 12 14

OD

60

0n

m

Time (hrs.)

Growth Curve of Uninduced E.coli BL21(DE3)

OD600nm

5. Acetate Production

• Glycerol levels rapidly decrease during the process with a concomitant

accumulation of acetate to approximately 5 g/l and a decrease of pH.

• At low glycerol concentrations the cells switch to a utilisation of the accumulated

acetate with an accompanying pH increase. This accumulated acetate provides the

carbon source during recombinant protein production.

• As little as 1 g/L acetate has a bacteriostatic effect while concentrations higher

than 4 g/L have a bactericidal effect on the host under the conditions used.

0,1

1

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100

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0 2 4 6 8 10 12 14 16 18 20 22 24 26

OD

60

0n

m

Co

nce

ntr

atio

n (

g/L)

Time (hrs) Glycerol Acetate pH OD600nm

0,1

1

10

0 2 4 6 8 10 12

OD

60

0n

m

Time (hrs.)

TB 0.5 g/l HOAc 1.0 g/l HOAc 1.5 g/l HOAc 2 g/l HOAc

3 g/l HOAc 4 g/l HOAc 5 g/l HOAc 6 g/l HOAc

7. Plasmid Stability

• Induction with IPTG leads to rapid loss of

plasmid.

• Plasmid stability remains at maximum for the

duration of cultivation in the absence of

induction.

• Induction at the later stages of the growth

curve leads to slower loss of plasmid.

0

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80

100

0 2 4 6 8 10 12 14 16 18 20

% p

lasm

id b

ear

ing

cells

Incubation Time (hrs.)

2h 4h 8h Non Induced Cultures

Fig. 6: Monitoring of glycerol (HPLC), acetate

(HPLC) and biomass levels (OD600nm) as well as

pH as a function of incubation time using the

optimised process conditions for SELP production.

The vertical line at 8 hours marks the point of

induction with 0.5mM IPTG.

Fig. 7 - Effect of acetic acid , added at 0 hours, on

growth of E. coli BL21 DE3(+)/pET25b/SELP3

under the optimised shake flask conditions of the

present study.

6. Selection Agent Concentration

• The ampicillin concentration rapidly decreases

during the first hour of cultivation and is

already depleted at a culture OD600nm of

approximately 0.2.

• β-lactamase encoded on the pET expression

vector used leads to the observed degradation of

the selection agent.

• Productions without the use of a selection agent

have allowed for similar SELP production

levels to those with ampicillin.

Fig. 1: Media Optimisation. SELP production,

expressed as a percentage of the maximum, biomass

production (OD600nm) and minimum pH measured, as

a function of the culture medium used. SELP

production levels evaluated by SDS-PAGE.

Fig. 2: Effect of temperature on biomass and SELP

production. The right hand side of the curve (25-30, 25-

37, 30-37, 37-30, 37-42) represent the temperature shift

experiments: the initial temperatures used and the

temperatures used after induction.

Fig. 5 - Effect of induction time and induction period on

biomass and SELP production.

SELP and biomass production as a function of IPTG induction time

(2-14 hrs. incubation) and induction period (2, 4 and 6 hrs.) (top).

Growth curve of uninduced E.coli BL21(DE3) for comparison of

the induction time with the stage of growth (bottom).

Fig. 8: Ampicillin concentration (measured by the Kirby Bauer

assay) and biomass levels as a function of incubation time Fig. 8: Plasmid stability, with and without induction, at various

time points during the cultivation.

Cultures induced at 2, 4 and 8 hours (as indicated by the arrow) as

well as non-induced cultures are compared.

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OD

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0n

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Am

pic

illin

Co

nce

ntr

atio

n (

µg/

ml)

Incubation Time (mins.) Amp. Concentration OD600nm

This work was financed by the European Commission via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009-SME-3, collaborative project number 246176).